Brain responses to strong desire to void with synchronous urodynamics in healthy adults: a functional magnetic resonance imaging study based on graph theory

Abstract


Background
There are two primary functions for the lower urinary tract, namely storage and voiding.Humans can shift back and forth between the two phases, depend on the coordination of the brain, spinal cord pathway, peripheral nerve, bladder, urinary sphincter, and pelvic oor muscles.Signi cantly, any interruptions in functional coordination could lead to lower urinary tract dysfunctions (LUTD), manifesting symptoms such as urinary frequency, urgency, incontinence, and dysuria [1].We implored the urodynamic tests, a well-developed and applied urological clinical pro le to assess LUTD.Urodynamic tests [2,3,4] was considered an ideal method to estimate both storage and voiding function with a speci c transurethral and a transrectal catheters.A strong desire to void is characterized by a quiet crucial sensory parameter tightly related to urinary continence [5]; additionally, it impacts the gait of individuals with multiple sclerosis and urinary disorders and [6,7].As reported in a study by Kaynar et al. [8], the highest maximum ow rate and average ow rate are obtained with a strong desire to void, suggesting its in uences on the urinary voiding.
Presently, functional magnetic resonance imaging (fMRI) based on blood oxygen level-dependent (BOLD) is being applied to investigate the supraspinal control of the lower urinary tract (LUT) in healthy subjects and patients with LUTD.Likewise, activations in the dorsal pontine, insula, prefrontal cortex, supplementary motor area, and the anterior and the middle cingulate gyrus have been found in healthy subjects [9].Abnormally activated brain regions and the alternations of functional connections in diseases with LUTD have been reported, including overactive bladder [10,11], stroke, and multiple sclerosis [12].Nonetheless, the topologic property changes were barely known.Although different patterns (especially the perfusion speed) of urinary bladder lling were generally considered as the potential in uencing factors for the result of urodynamic results, the alternations of brain responses resulting from these patterns were unclear yet.
A series of baseline brain fMRI study concerning a strong desire to void was launched in healthy subjects to determine the central mechanism of the various LUTD in future.In the series, various patterns of urinary bladder lling were involved.In the present article, with a repetitive infusion and withdrawal pattern, the functional brain network's topologic property alternations evoked by a strong desire to void in healthy male and female adults will be respectively reported.

Participants and Ethics Statement
Twenty-two healthy participants (11 males and 11 females) between 2017 and 2018 were enrolled.All healthy participants were asked to ll a 3day voiding diary.Notably, the physical tests were satisfactory, and no abnormal medical history existed.The 24-hour urine volume ranged between 1500 to 3000ml.(5) tumors, especially pelvic tumors; (6) currently undergoing radiotherapy or chemotherapy; (7) taking medications or other substance which could impact on the nervous system or urinary system; (8); pregnancy test was positive; (9) claustrophobia; and (10) having medical taboo to scanning.These participants signed an informed consent form and the study was executed in accordance with the revised Helsinki Declaration, and the study's ethics approval was provided by the Ethics committee of China Rehabilitation Research Center (IRB: 2017-002-1).

Image acquisition and preprocessing
All images were acquired on an Ingenia 3.0 Tesla scanner (Philips, Eindhoven, The Netherlands).Before fMRI scans, the subjects were asked to void.Subjects lay supine while wearing headphones.A xed device was used to limit the motion of patient's head.A 7 Fr double-lumen bladder catheter and a rectal catheter were inserted to monitor vesical pressure and intra-abdominal pressure with a portable urodynamic device (Laborie Medical Technologies, Vermont, United States).The procedure's complete details were explained to the subjects, including closing their eyes and lying awake during scanning without any systematic thoughts.Structural T1 images (3D; repetition time = 7.8ms; echo time = 3.8 ms; and ip angle = 8 degrees) were collected 10mins after voiding.Then a gradient-echo, echo-plane imaging sequence (echo time = 30ms; repetition time = 2000ms; ip angle = 90 degrees; voxel size = 3× 3× 3.5 mm) was applied to all volunteers' rst resting-state fMRI scans.
The scanning lasted 6mins 14sec, and synchronous urodynamics evaluation was performed during the rst resting-state functional brain scanning.After 200ml of 0.9%, sterile saline solution was infused into the subjects' bladder by a syringe; the urinary bladder was lled via four blocks (Fig. 1) with sterile saline solution.The second resting-state for fMRI scans were performed using prior resting-state parameters, while a strong desire to void was reported.The urge to void was evaluated using a visual analog scale (score 0: empty bladder; score 6: strong desire to void; score 8: urge desire to void; score 10: pain), which had been applied in previous researches [13,14].Finally, bladder volume was measured after the voluntary elimination into a counting cup.
Standard preprocessing of acquisitions was performed with SPM8 (https://www.l.ion.ucl.ac.uk/spm/software/spm8).The rst ten volumes were excluded from functional scans for magnetization equilibration.Slice timing correction, motion correction, the co-register between functional and structural scans, segmentation of structural images, normalization to Montreal Neurological Institute space, resampling with 3mm×3mm×3mm voxel size, spatial smoothing with a 6mm Gaussian kernel, detrending, and ltering with low-pass frequency lter (0.01-0.1Hz) were performed step by step.The data with head horizontal displacement in three-dimensional space > 2 mm and head rotation > 2° would be excluded during motion correction.Finally, nuisance signals resulting from head motion, white matter, cerebrospinal, and the global signal were regressed out.

Establishment of connection matrix/networks
The functional brain networks were established via graph theory methods, where nodes represent various brain regions, while edges were modeled as a pairwise connection between nodes.The graph theory analysis was performed using GRETNA 2.0.0 toolbox (https://www.nitrc.org/projects/gretna/).Reign of interest (ROI), i.e., brain nodes, were initially identi ed using the automated anatomical labeling (AAL) atlas.The brain was divided into ninety anatomic regions (regions 1-90) using the ALL atlas.Then the time series of the corresponding brain regions were extracted from the preprocessed data.Pearson's correlation calculations were performed to determine the associations between achieving the time series of pairwise different brain regions.Furthermore, a 90 × 90 brain connection matrix (Fig. 2) was achieved from each subject's data.For normalization, a z-score was calculated with the Fisher r-to-z method.The graph analysis irrelevant or weak functional connections were excluded with proportional network thresholding (sparsity T, 0.05-0.5;intervals, 0.05).When Pearson's correlation coe cient of individual pairs of connections was not greater than T, the corresponding connections were identi ed and removed.Otherwise, the functional connections were considered to exist.The thresholding methods had been reported in the previous study [15].

Graph analysis-Network Property Analysis
The twenty-two subjects were divided into the healthy male group and the healthy female group.Smallworld properties, global e ciency (E glob ), and local e ciency (E loc ) were analyzed for the operational data after voiding and the acquired functional scans when subjects strongly desired voiding.The characteristic path length (L p ) and the clustering coe cient (C p ) are signi cant global metrics.L p , a parameter estimating the network's integration function, is de ned as the average shortest path lengths for all possible pairs of nodes.C p expresses the density of connection of a node's connection, which measures the network's segregation.The more outstanding C p is, the denser the connections are.High E glob and E loc in the small-world network ensure the optimal function in segregation and integration.C p and L p of the entire functional brain networks were normalized via matching with generated random networks (n = 100) for the quantity of the small-world characteristics.
The gamma (γ) and lambda (λ) were obtained from the L p and C p of the entire network and random networks (γ = C p real / C prand ; λ = L p real /L p rand ).Sigma (σ, σ = γ/λ) was used to describe the small-world coe cient.If γ > 1, λ ≈ one, and σ > 1, the network has small-world properties.E glob was used to estimate the network's communication e ciency and E loc expressed the e ciency of the local subgraph of speci c nodes that contains only the direct neighboring nodes.In terms of regional network properties, nodal e ciency (E nodal ) evaluating a given node's capacity for information communication with the other nodes was calculated.

Statistical analyses
For statistical analysis, a paired t-test (P < 0.05) and Bonferroni correction between the empty bladder and strong desire to void was applied in both groups to obtain signi cant differences in small-world topologic property parameters (γ and σ), L p , C p , E glob , E loc, and regional E nodal .

Results
The resting state fMRI data of eleven males and eleven females were analyzed in the study.A male and a female were excluded for head motion (head displacement > 2 mm and head rotation > 2°  1).There wasn't statistical difference between the both group in the sensory score of visual analog scale.Urodynamic ndings showed the stationary and normal urinary storage in all healthy subjects, without detrusor overactivity detected (Fig. 3).Adverse events after the study Frequency/Urgency/Leakage(times) 0 0 Dysuria/Urinary retention/hematuria 0 0

Global functional network properties
After normalizing with random networks, higher C p (γ > 1) and L p (λ ≈ 1) for network sparsity was observed in all subjects under the empty bladder (mean ± SD, γ in female group:1.48± 1.95; γ in the male group: 2.00 ± 2.25; λ in the female group: 0.30 ± 1.14; λ in the male group: 0.31 ± 1.14) and strong desire to void state (mean ± SD, γ in the female group:1.52 ± 2.01; γ in male group:1.55 ± 2.03; λ in female group:0.23 ± 1.10; λ in male group: 0.22 ± 1.10).Small-world properties (σ > 1) was detected in both groups under empty bladder (mean ± SD, σ in the female group: 0.63 ± 1.59; σ in the male group: 0.94 ± 1.81) and strong desire to void state (mean ± SD, σ in the female group: 0.77 ± 1.70; σ in the male group: 0.86 ± 1.73).There were no signi cant differences in the small-world coe cient (σ, P > 0.05) between the states in the two groups.Compared with the empty bladder state, the signi cant decreased C p , L p , E loc , and increased E glob (P 0.05) were detected in the female group under a strong desire to void (Fig. 4).In the male group, signi cant decreased E loc in the state with a strong desire to void.There were no statistical differences between the two states in C p , Lp, and E glob .(P > 0.05) (Fig. 5).
Notably, a signi cant increase in E nodal (P < 0.05 after FDR correction) in the state with a strong desire to void was detected in the right frontal operculum (Rolandic_Oper_R), left supplementary motor area (Supp_Motor_Area_L), medial superior frontal gyrus (Frontal_Sup_Medial_R), and the bilateral supramarginal gyrus (SupraMarginal_L and SupraMarginal_R).However, a decrease in E nodal was observed in the inferior occipital gyrus (Occipital_Inf_R) and thalamus (Thalamus_L) (Fig. 6c, Fig. 6d).

Discussion
In the past two decades, more than twenty studies addressed brain fMRI ndings on urinary bladder control while simultaneous healthy females and males were barely enrolled as subjects or healthy controls in these studies [16].In the study, the data of the healthy female group and male group were obtained and analyzed under the same scanning parameters, respectively.The differences of brain topologic property alternations evoked by the strong desire to void state may provide the understanding for the central-LUT control mechanism in healthy women and men.

Global graph metrics
In the study, the repetitive infusion and withdrawal pattern was used to activate the regions related to LUT control.Small-world network properties were observed in the empty bladder and a strong desire to void state in both groups.High C p and low L p are the small-world architecture's outstanding characteristics, which are optimized for information processing.Balanced functional integration and segregation were observed in the small-world architecture according to previous general assumptions.High E glob and E loc were detected in the small-world networks, which demonstrated higher e ciency in global and local information communication than the regular network (with low E glob and high E loc ) and the random network (with high E glob and low E loc ).
Although the small-world properties were detected in both states, the signi cantly decreased C p and E loc were observed in the females provoked by a strong desire to void compared with the empty bladder state, which revealed the lower capacity in local information processing and the decreased e ciency in local information transmission.The signi cantly decreased L p and increased E glob in globally connected graphs suggested the higher capacity in the information processing and higher e ciency in global information communication transmission and a better functional integration in female group.
In the male group, the decreased E loc was observed under the strong desire to void state compared with the empty bladder state, revealing the lower e ciency in local information transmission.The results implied the decreased trendy of functional segregation in the male group.There were no statistical differences of C p , L p and E glob between the both states.

Regional nodal metrics
In the female group, the signi cant increased E nodal under a strong desire to void state was detected in left inferior frontal gyrus and the orbital part of middle frontal gyrus, right median cingulate gyrus, middle occipital gyrus and middle temporal gyrus, and bilateral gyrus rectus, inferior parietal gyrus and supramarginal gyrus.In the male group, the increased E nodal presented in right frontal operculum and medial superior frontal gyrus, left supplementary motor area and the bilateral supramarginal gyrus.The signi cant decreased E nodal in female group was detected in the bilateral calcarine ssure and surrounding cortex, lingual gyrus, and fusiform gyrus.The decreased E nodal in male group presented in right inferior occipital gyrus and thalamus.
Present clinical human trials and animal experiments [17,18,19] have showed that there was a notable voiding re ex between the bladder and the midbrain periaqueductal gray (PAG).During the bladder storage, the afferent lling sensory signals resulted in bladder distension until the volume threshold in PAG was exceeded.The voiding re ex was provoked to relax the urethral sphincter and contract bladder detrusor, while voiding.Urinary bladder storage restarted when it was empty.In fact, the higher central mechanism works in the entire bladder lling and voiding process.
The prefrontal cortex (PFC) is crucial for the LUT control.PFC involved in human personality, decisionmaking, and social behavior.Signi cantly, the cortex has been presumed to control voluntary action, including deciding to void [20].In a previous research [21], as the urinary bladders were passively infusion, heathy female brain responses to larger bladder volume increased in the orbitofrontal cortex.But in patients with overactivity bladder and poor bladder control had weaker responses in the region.The lateral PFC concerns cognition, especially in work memory [22].Structural MRI has demonstrated the failure to postpone voiding due to the lateral PFC lesion in adult males and females [23].Children with monosymptomatic nocturnal enuresis manifested abnormal resting-state connectivity in the region [24].
In the study, the regional E nodal in lateral PFC where left opercular part of inferior frontal gyrus located indeed increased compared with the empty bladder in female group.Early Positron-emission tomography (PET) and single photon emission computed tomography (SPECT) studies reported the activation in the bilateral inferior frontal gyrus, but the evidences were absent in male fMRI [9] and the increased E nodal only presented in female subjects in the study.An increased regional E nodal at medial PFC was detected in both groups.Medial PFC was an essential part of default mode network (DMN) [25].Interoceptive and spatial representations of the body were integrated into DNM, including the bladder sensory [26].When it came to self-awareness and self-re ection under a resting state, DNM was activated.Additionally, the region works in the cognitive process, regulating emotion, and sociability [27].Medial prefrontal gyrus lesions were found to result in relatively short-term incontinence in adults.Nevertheless, the white-matter lesion in the medial PFC also led to long-term urinary bladder dysfunction [28].And a fMRI research showed the region was deactivated under the full urinary bladder in patients with urgency incontinence [29].The gyrus rectus was an essential region of the medial prefrontal network, which mediated the interaction between the visceromotor centers and the prefrontal sensory signals via the hypothalamus's descending pathway and the brainstem [30].The ventromedial PFC was proved to connect with the limbic system and other brain regions, which determine its vital roles in LUT control.
The cingulate gyrus as a component of the limbic system was known for multiple functions such as the mediation of emotional and autonomic responses to external stimuli, and processing the information from the bladder to maintain urinary continence and impact on the urge to void [31,32].The cingulate gyrus was involved in visceral stimulation and the urge to void was considered as a nonpainful visceral stimulation [11].As mentioned, the anterior or median cingulate cortex controls the heart rate via sympathetic mechanisms [33,34].The dorsal anterior or median cingulate cortex was speculated to control the lower urinary tract via the same mechanism, which cannot be precisely identi ed now.Under a strong desire to void or urgency, the activated dorsal anterior cingulate cortex would facilitate to urinary continence by urinary sphincter contraction and bladder relaxation [35] .In our study, we detected an increased E nodal in right median cingulate gyrus under the strong desire to void.
Pelvic oor muscles which are important in stress urinary incontinence cannot be isolated.In the male group, supplementary motor area (SMA),an adjacent location to dorsal anterior cingulate cortex, showed the increased E nodal under the strong desire to void.In previous researches, SMA showed the activated response during the pelvic oor muscle's voluntary contraction [36,37].Yin et al. demonstrated that the middle temporal gyrus and the right inferior frontal gyrus inhibited detrusor contraction together during urinary bladder storage in healthy subjects [38].Patients with detrusor overactivity were found weaker in the two regions [36].Besides Cohen and coworker [39] suggested that the cingulate gyrus and premotor cortex played important roles in the regulating selective attention under task-con ict condition.During the research, these subjects who had the strong desire to void knew voiding in the scanner was inappropriate.
Visceral perception, including urinary bladder lling sensory, can be integrated with exteroceptive and interoceptive signals [14].The frontal operculum is a region adjacent to the insula,which involves the awareness and processing of interoceptive signals [37].The supramarginal gyrus, located in the inferior parietal lobe (IPL), showed a crucial association with proprioception.A recent study has suggested that the regions were activated under the condition of visceral perception, such as heartbeat [40].The Multifunction of IPL has been revealed in multisensory integration, spatial attention, higher cognitive functions, and oculomotor control [41,42].An earlier PET and fMRI study showed the IPL also responds to urinary bladder cooling during the bladder storage using ice water [ 13 43].Meanwhile, the human fusiform gyrus is a region concerning objects recognizing and functional de nition, which often interacts with the occipital lobe on visual tasks [44].The occipital cortex change has been mentioned in previous researches, but the mechanism was not systematically discussed [45,46].Distinctly, regional E nodal in the male group's thalamus was decreased under a strong desire to void state.
No difference was observed between urinary bladder lling and emptying in the female group.As a relay station, the thalamus transmits the sensory signals from PAG to the insula, lateral PFC and medial PFC in terms of a frame established via several animal and human researches [9,47].Decreased E nodal in thalamus suggesting the lower e ciency in information transmission in regionally connected graphs.
The study of Kuhtz-Buschbeck et al [35] had similar gender differences, in which thalamus in healthy males was less activated under the urge to void compared with healthy females.But the activity in other brain regions under the both urinary bladder states were not compared in detail.In our study, compared with the empty urinary bladder state, the more regions of PFC in female group showed the increased E nodal under a strong desire to void state.SMA as the motor control was signi cantly with the increased E nodal in the male group but not in the female group.Gehring and Knight [48] had suggested the activation in premotor cortex worked with the cingulate gyrus to monitor behavior and guiding compensatory system, the gender difference may partly result from the compensatory system.Under a strong desire to void, subjects' brain monitored and regarded that the voiding in the scanner was inappropriate, the pelvic oor muscle contraction might be initiated as a compensatory mechanism to resist urine leakage.
No consensus has been reached in gender differences in central LUT control.Compared with female genitourinary system, the male longer urethra and prostate may impact on the brain response for the desire to void with or without the catheters.Blok and his coworkers [49] had indicated that different level of activation in the insula, hypothalamus and PAG during micturition and urine withholding between healthy female and males in his PET study.A meta-analysis of neuroimaging studies has revealed that activated clusters in brainstem (periaqueductal gray and rostral pons), thalamus, insula, and cerebellum to response the urinary bladder lling and no signi cant difference in brain activation between female and male subjects was detected [50].Although only cortical and subcortical regions without pons and cerebellum were involved in our study, its results have provided the evidence for gender difference in responses to the strong desire to void under the pattern.
Finally, the sample size was small, which may be a potential limitation of the study.On the other hand, due to the AAL atlas we used to de ne the regions of interest, only brain activity in cortical and subcortical regions were in focus.We will consider the future studies covering the cerebellum and pons to obtain more integrated information.

Conclusions
With the repetitive infusion and withdrawal pattern, we detected different functional topologic property alternations of the healthy female and male subjects between the strong desire to void and empty bladder state in our study.The baseline ndings in healthy females and males might help understand the underlying pathogenesis in LUTD patients during the urodynamic tests.Brain functional connection matrix of healthy female and male subjects under the both of states.
Page 18/21    The brain regions with an alternated Enodal in the healthy male subjects between the both of states.
Urination frequency was eight times/day and ≤ one time/night with an average urine volume of 200ml/time.No urine leakage.The exclusion criteria included having: (1) LUT symptoms; (2) neurological diseases or lesion, such as myelitis; (3) multiple sclerosis; (4) traumatic spinal cord injury;

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Figure 4 The
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Figure 6
Figure 6 Legend: (a) For female subjects, a larger Enodal in bilateral calcarine ssure and surrounding cortex, lingual gyrus, and fusiform gyrus under empty bladder state.(b) Under the strong desire to void, a larger Enodal in left inferior frontal gyrus and the orbital part of middle frontal gyrus, right median cingulate, middle occipital gyrus and middle temporal gyrus, and bilateral gyrus rectus, inferior parietal gyrus and supramarginal gyrus was detected in healthy females.(c) The larger Enodal in right inferior occipital gyrus and thalamus of male subjects under empty bladder state.(d) The signi cant increased Enodal of healthy male subjects presented in right frontal operculum and medial superior frontal gyrus, left supplementary motor area and the bilateral supramarginal gyrus (P 0.05).